Greenhouse gas (GHG) emissions from rivers and lakes have been shown to contribute significantly to global carbon and nitrogen cycling. In temperate and human-impacted regions, simultaneous carbon dioxide, methane and nitrous oxide emissions from aquatic systems are poorly documented. We estimated carbon dioxide (CO2) concentrations in the Seine hydrosystem (71,730 km2, France) using direct measurements, and calculations of CO2 partial pressures from 14 field campaigns conducted between 2010 and 2017, and compared them to methane (CH4) and nitrous oxide (N2O) concentrations. In the main stem of the Seine River, CO2 showed the same spatial gradient as N2O and CH4 with peaks in concentration downstream from the arrival of effluents from wastewater treatment plants enriched in organic matter, thus favoring mineralization. It is likely that high CO2 concentrations upstream were due to organic carbon inputs from soils and enriched CO2 groundwater discharges, whereas high N2O and CH4 upstream values were likely due to denitrification in riparian wet areas and anoxic decomposition of organic matter-rich wetlands, respectively. In addition, seasonal variations in all three GHGs were observed with higher concentrations in summer when higher temperatures promote mineralization and low water reduces the dilution of organic matter mainly originating from WWTP effluents. GHG emissions were calculated and compared with agricultural and nonagricultural (urban, transport) fluxes in the basin. In the Seine River network, CO2 emissions dominated riverine GHG emissions, reaching 95.3%, while N2O and CH4 emissions accounted for 4.4% and 0.3%, respectively. These indirect emissions from the hydrosystem were estimated to account for 3.7% of the total GHG emissions from the basin that amounted to 61,284 Gg CO2eq yr−1. Comparatively, direct agricultural and nonagricultural GHG emissions were estimated at 23.3% and 73.0%., respectively.